Methods have been previously described to incorporate unnatural amino acids site-specifically into proteins in mammalian cells. Chemically aminoacylated suppressor tRNAs have been microinjected or electroporated into CHO cells and neurons, respectively, and used to suppress nonsense amber mutations with a series of unnatural amino acids (Monahan et al. (2003), “Site-specific incorporation of unnatural amino acids into receptors expressed in Mammalian cells,” Chem Biol 10:573-580). However, the use of the aminoacylated tRNA as a stoichiometric reagent severely limits the amount of protein that can be produced.
Alternatively, heterologous suppressor tRNA/aaRS pairs that do not cross react with host tRNAs, aaRSs or amino acids (orthogonal tRNA/aaRSs) have been engineered to incorporate unnatural amino acids selectively into proteins. For example, Yokoyama and coworkers modified a Bacillus stearothermophilus amber suppressor tRNACUATyr (BstRNACUATyr) and E. coli tyrosyl-tRNA synthetase (EcTyrRS) to incorporate 3-iodo-L-tyrosine into proteins in CHO cells (Sakamoto et al. (2002), “Site-specific incorporation of an unnatural amino acid into proteins in mammalian cells,” Nucleic Acids Res 30:4692-4699). Similarly, Zhang and coworkers engineered an orthogonal Bacillus subtilis suppressor tRNA/tryptophanyl-tRNA synthetase pair to incorporate 5-hydroxytryptophan into proteins in mammalian cells with high fidelity (Zhang et al. (2004), “Selective incorporation of 5-hydroxytryptophan into proteins in mammalian cells,” Proc Natl Acad Sci USA 101:8882-8887).
However, the use of structure-based mutagenesis to generate aaRS variants that aminoacylate an amino acid whose side chain differs significantly from that of the wild type substrate requires mutations of multiple active site residues which are difficult to predict a priori (Zhang et al. (2002), “Structure-based design of mutant Methanococcus jannaschii tyrosyl-tRNA synthetase for incorporation of O-methyl-L-tyrosine,” Proc Natl Acad Sci USA 99:6579-6584; Turner et al. (2005), “Structural characterization of a p-acetylphenylalanyl aminoacyl-tRNA synthetase,” J Am Chem Soc 127:14976-14977; Turner et al. (2006), “Structural plasticity of an aminoacyl-tRNA synthetase active site,” Proc Natl Acad Sci USA 103:6483-6488). Moreover, the engineered mutant may still recognize host amino acids, as is the case with a mutant aaRS that charges its cognate tRNACUATyr with 3-iodo-L-tyrosine (Sakamoto et al. (2002), “Site-specific incorporation of an unnatural amino acid into proteins in mammalian cells,” Nucleic Acids Res 30:4692-4699; and Kiga et al. (2002), “An engineered Escherichia coli tyrosyl-tRNA synthetase for site-specific incorporation of an unnatural amino acid into proteins in eukaryotic translation and its application in a wheat germ cell-free system,” Proc Natl Acad Sci USA 99:9715-9720).
Alternatively, one can attempt to evolve aaRSs with altered specificities directly in mammalian cells. For example, Wang and coworkers recently used somatic hypermutation in a human B cell line to directly evolve a monomeric red fluorescent protein with enhanced photostability and far-red emissions (Wang et al. (2004), “Evolution of new nonantibody proteins via iterative somatic hypermutation,” Proc Natl Acad Sci USA 101:16745-16749). However, somatic hypermutation introduces random mutations in the whole protein, which may be less effective than genetic diversity created by targeted mutagenesis of the active site when evolving variants with altered substrate specificity. The latter, however, is limited by difficulties in generating large stable libraries in mammalian cells.
Orthogonal Translation Technology
A general methodology has been developed for the in vivo site-specific incorporation of structurally diverse unnatural amino acids with non-native physical, chemical and biological properties into proteins in prokaryotic organisms and yeast. These methods rely on orthogonal protein translation components that recognize a suitable selector codon to insert a desired unnatural amino acid at a defined position in a gene of interest during polypeptide translation in vivo. These methods utilize an orthogonal tRNA (O-tRNA) that recognizes a selector codon (e.g., a nonsense amber codon), and where a corresponding specific orthogonal aminoacyl-tRNA synthetase (an O-RS) specifically charges the O-tRNA with the unnatural amino acid. These components do not cross-react with any of the endogenous tRNAs or RSs in the host organism (i.e., the engineered tRNA and RS are orthogonal).
Using this technique in E. coli host systems, functional amber and frameshift suppressor tRNA/aaRS pairs have been derived from a Methanococcus jannaschii tRNATyr/TyrRS pair, an archaeal tRNAGlu/Pyrococcus horikoshii glutamyl-tRNA synthetase pair, and an archaeal tRNALys/Pyrococcus horikoshii lysyl-tRNA synthetase pair. In Saccharomyces cerevisiae (S. cerevisiae), functional tRNACUA/aaRS pairs have been derived from the corresponding E. coli tRNATyr/TyrRS and tRNALeu/leucyl-tRNA synthetase pairs. Directed evolution of these suppressor tRNA/aaRS pairs using a combination of positive and negative selections has allowed the efficient, highly selective in vivo incorporation of a large number of diverse unnatural amino acids in E. coli and S. cerevisiae. These include fluorescent, glycosylated, sulfated, metal-ion-binding, and redox-active amino acids, as well as amino acids with novel chemical and photochemical reactivity. This methodology provides a powerful tool for exploring protein structure and function in vitro and in vivo, and for generating proteins, e.g., therapeutic proteins, with new or enhanced properties. The extension of this methodology to mammalian cells, for example primate and rodent cell lines, would significantly enhance the utility of this technology.
The practice of using orthogonal translation systems that are suitable for in vivo production of proteins that comprise one or more unnatural amino acid is generally known in the art. For example, see International Publication Numbers WO 2002/086075, entitled “METHODS AND COMPOSITION FOR THE PRODUCTION OF ORTHOGONAL tRNA-AMINOACYL-tRNA SYNTHETASE PAIRS;” WO 2002/085923, entitled “IN VIVO INCORPORATION OF UNNATURAL AMINO ACIDS;” WO 2004/094593, entitled “EXPANDING THE EUKARYOTIC GENETIC CODE;” WO 2005/019415, filed Jul. 7, 2004; WO 2005/007870, filed Jul. 7, 2004; WO 2005/007624, filed Jul. 7, 2004 and WO 2006/110182, filed Oct. 27, 2005, entitled “ORTHOGONAL TRANSLATION COMPONENTS FOR THE VIVO INCORPORATION OF UNNATURAL AMINO ACIDS.” Each of these applications is incorporated herein by reference in its entirety.
Additional discussion of orthogonal translation systems is also found in, for example, Wang et al. (2001), “Expanding the genetic code of Escherichia coli,” Science 292:498-500; Wang and Schultz (2002), “Expanding the Genetic Code,” Chem. Commun. (Camb.) 1:1-11; Alfonta et al. (2003), “Site-Specific Incorporation of a Redox-Active Amino Acid into Proteins,” J Am Chem Soc 125:14662-14663; Santoro et al. (2003), “An archaebacteria-derived glutamyl-tRNA synthetase and tRNA pair for unnatural amino acid mutagenesis of proteins in Escherichia coli,” Nucleic Acids Res 31:6700-6709; Chin et al. (2003), “An expanded eukaryotic genetic code,” Science 301, 964-967; Chin et al. (2003), “Progress toward an expanded eukaryotic genetic code,” Chem Biol 10, 511-519; and Wu et al. (2004), “A genetically encoded photocaged amino acid,” J Am Chem Soc 126, 14306-14307; Summerer et al. (2006), “A Genetically Encoded Fluorescent Amino Acid,” PNAS 103(26):9785-9789; Anderson et al. (2004), “An expanded genetic code with a functional quadruplet codon,” Proc Natl Acad Sci USA 101:7566-7571; Zhang et al. (2004), “A new strategy for the synthesis of glycoproteins,” Science 303, 371-373; Wang and Schultz “Expanding the Genetic Code,” Angewandte Chemie Int. Ed., 44(1):34-66 (2005); Xie and Schultz, “An Expanding Genetic Code,” Methods 36(3):227-238 (2005); Xie and Schultz, “Adding Amino Acids to the Genetic Repertoire,” Curr. Opinion in Chemical Biology 9(6):548-554 (2005); Wang et al., “Expanding the Genetic Code,” Annu. Rev. Biophys. Biomol. Struct., 35:225-249 (2006); Xie and Schultz (2006), “A Chemical Toolkit for Proteins—an Expanded Genetic Code,” Nat. Rev. Mol. Cell. Biol., 7(10):775-782; Summerer et al. (2006), “A Genetically Encoded Fluorescent Amino Acid,” Proc Natl Acad Sci USA 103, 9785-9789; Wang et al. (2006), “A Genetically Encoded Fluorescent Amino Acid,” J Am Chem Soc 128, 8738-8739; and Liu and Schultz (2006), “Recombinant Expression of Selectively Sulfated Proteins in E. coli,” Nat. Biotechnol., 24(11): 1436-1440. The content of each of these publications above is hereby incorporated by reference.
There is a need in the art for the development of improved orthogonal translation components that incorporate unnatural amino acids into proteins in mammalian cell host systems, for example in primate and rodent host cell systems, where a desired unnatural amino acid is incorporated at defined positions. There is a need in the art for improved methods for screening and identifying orthogonal translation components (e.g., mutant aminoacyl-tRNA synthetase enzymes) that can function in mammalian cells, such as rodent cells and human cells. The invention described herein fulfills these and other needs, as will be apparent upon review of the following disclosure.